This invention relates to novel polypeptides, bacteriocins, immunity genes obtained from lactic acid bacteria and a method of use thereof.
With the current consumer demand for fresh (i.e., never frozen) foods, it is important that methods be developed for safe storage of these products especially for fresh meats which are manufactured locally but are marketed around the world. The lactic microflora (lactic acid bacteria) of vacuum packaged meats delays spoilage for weeks or months, as opposed to meats packaged under aerobic conditions which develop a putrefactive microflora that causes spoilage within days.
Vacuum packaged meats have an extended but unpredictable storage life dependent on the types of Tactics that dominate the microflora. Meat Tactics can cause severe spoilage problems, such as sulphide odors or greening by some Lactobacillus species and gas or slime production by Leuconostoc species. Other Tactics exert a preservative effect, extending storage life and enhancing meat safety by competitive growth, by producing organic acids, and by producing antagonistic substances known as bacteriocins (peptides or proteins that inhibit the growth of other, usually closely related, bacteria).
Nisin is a bacteriocin produced by lactics used for cheese manufacture, and is the only bacteriocin licensed for use as a food preservative. Nisin is unusual because it is active against a wide range of gram-positive bacteria, including the spores of Clostridium botulinum; unfortunately, its producer strain does not grow in chill-stored meats, and nisin does not function in meat systems.
Class II bacteriocins are characterized as small, heat stable, hydrophobic peptides with a high isoelectric point. They are produced as precursors with an N-terminal extension of 18 to 24 amino acids. This extension is cleaved at the C-terminus side of two glycine residues to give the mature bacteriocin. Sequence alignment of the N-termini revealed a remarkable degree of similarity in their hydropathic profiles (Fremaux et al. 1993).
The nucleotide sequences of the structural genes for several class II bacteriocins have been published, including pediocin PA-1/AcH (Bukhtiyarova et al. 1994, Marugg et al. 1992), sakacin A and P (Holck et al. 1989, Tichaczek et al. 1994), lactacin F (Fremaux et al. 1993, Muriana and Klaenhammer 1991), leucocin A (Hastings et al. 1991), lactococcins A, B, and M (Holo et al. 1991; Stoddard et al. 1992; van Belkum et al. 1991; van Belkum et al. 1992), plantaricin A (Diep et al. 1994) and carnobacteriocins A, BM1, and B2 (Quadri et al. 1994; Worobo et al. 1994). However, the additional genes necessary for bacteriocin production have only been determined for the lactococcins and pediocin PA-1/AcH and, in the case of the some of the lactococcins, the gene for immunity has also been confirmed. The genetic characterization of the lactococcin and pediocin gene clusters indicates that they have similar features. They both have genes for bacteriocin production in an operon structure, although the structural and immunity genes for the lactococcins can be transcribed independent of the other genes in the operon. Furthermore, one of the genes in each of the lactococcin and pediocin operons encodes a protein which belongs to the HlyB-family of ATP-binding cassette (ABC) transporters (Higgins 1992). This protein is thought to be involved in the signal sequence-independent secretion of the bacteriocins. Recently, genes encoding proteins which resemble members of a two-component signal transduction system have been identified which are involved in the expression of plantaricin A and sakacin A (Axelsson et al. 1993; Diep et al. 1994).
One aspect of the invention is a new bacteriocin, brochocin-C: peptide A (SEQ ID NO:23), peptide B (SEQ ID NO:25) and its corresponding immunity peptide (SEQ ID NO:27). Another aspect of the invention is a polynucleotide encoding the brochocin-C operon (SEQ ID NO:21), peptide A (SEQ ID NO:22), peptide B (SEQ ID NO:24), or immunity (SEQ ID NO:26).
Another aspect of the invention is a polynucleotide encoding a new bacteriocin enterocin 900 (SEQ ID NO:28), a polynucleotide encoding the first enterocin 900 peptide (SEQ ID NO:29), and the enterocin 900 peptide (SEQ ID NO:30).
Another aspect of the invention is a method for inhibiting pathogenic bacteria by providing a bacteriocin selected from the group consisting of brochocin-C and enterocin 900, either as a composition or by providing a bacterial source of brochocin-C or enterocin 900. For example, one may inhibit spoilage bacteria in foodstuffs, such as meat, inhibit pathogenic bacteria topically on animals, including humans, and inhibit bacteria infection of fermentation reactors.
Another aspect of the invention is an expression vector for obtaining secretion of proteins from lactics, comprising a promoter functional in the lactic host, a polynucleotide encoding a divergicin signal peptide (SEQ ID NO:19), and a structural gene. Another aspect of the invention is the vector which comprises a plurality of structural genes, each operably linked to a polynucleotide encoding a divergicin signal peptide.
Another aspect of the invention is a method to attach bacteriocin structural and immunity genes to a signal peptide or leader peptide gene so that the bacteriocins can be exported from the host cell.
Another aspect of the invention is a novel food-grade plasmid that can be used as a plasmid vector for genes including, but not limited to, bacteriocins, other polypeptides, enzymes or proteins in organisms for use in food products or as a probiotic.
Another aspect of the invention is a method to preserve food by adding bacteriocin-producing bacteria.